Mesenchymal stem cells (MSCs) are multipotent, and are capable of differentiating into more than one type of mesenchymal cell lineage, such as adipocytes, cardiac muscle cells and skeletal muscle cells (Galmiche et al., (1993) Blood 82:66-76; and Wakitani et al., (1995) Muscle Nerve 18:1417-1426). Thus, MSCs has gained considerable interest as a treatment for a myriad of diseases, conditions, and disabilities because they provide a renewable source of cells and tissues, and most importantly, unlike embryonic stem cells, mesenchymal stem cells pose no ethical dilemma in using them as a cell source.
Bone marrow mesenchymal stem cells (BMSCs) have been show shown to differentiate into cardiomyocyte-like cells by use of a DNA demethylating agent (e.g., 5-azacytidine) or by co-culturing with rodent cardiomyocytes. However, 5-azacytidine is cytotoxic and co-culturing with rodent cells runs the risk of causing xeno-contamination to the recipient of the cells. In this regard, improved methods have been proposed.
WO 2004/065589 A1 disclosed a method of producing cells for transplantation into myocardial tissue. According to Example 1 of '589 patent, BMSCs were first isolated and cultured in a medium containing 10% fetal bovine serum, 100 μM L-ascorbic acid-2-PO4, 5-15 ng/ml human LIF (leukemia inhibitory factor) and 20 nM dexamethasone. This medium allows the BMSCs to maintain their self-renewing character and to expand by passaging without losing responsiveness to the differentiation agents such as growth factors. Induction of BMSCs into cardiomyocytes were achieved by culturing BMSCs in the presence of growth factors (50 ng/ml bFGF and 25 ng/ml BMP-2) and IGF-1 (2 ng/ml) for 2 weeks.
WO 2005/056779 disclosed a method of producing cells for transplantation into myocardial tissue of a mammal comprising culturing bone marrow stem cells in a cardiac specific media contain bFGF, BMP-2 and IGF-1, and preferably, the media contain bFGF, BMP-2 and IGF-1 in each concentration of 1 to 200 ng/ml, and further contain 2 to 20% fetal bovine serum, 1 to 1000 μL-ascorbic acid-2-phosphate, 5 to 15 ng/ml leukemia inhibitory factor (LIF) and 1 to 200 nM dexamethasone.
U.S. Pat. No. 6,387,369 disclosed a method of producing cardiomycetes in vivo by administering to the heart of an individual, such as a human, a cardiomyocyte producing amount of mesenchymal stem cells. The MSCs were administered as a liquid injectable to the heart or as a preparation of cells in a matrix which is or becomes solid or semi-solid. The specification also teaches treatment of MSCs including the steps of treating MSCs with growth factors and differentiating agents as well as exposure of MSCs to mechanical stimuli and electrical stimulation, so that the treated MSCs may progress towards cardiomyocytes. However, it did not point out the exact species of suitable growth factors and/or differentiating agents.
Shim et al (Shim et al., (2004) BBRC 324:481-488) disclosed the use of cardiomyogenic differentiation medium containing insulin, dexamethasone and ascorbic acid. Differentiation was confirmed by the expression of cardiomyocyte-specific proteins such as cardio troponin I, sarcomeric tropomyosin and cardiac titin.
All of the above-identified publications teach differentiation of MSCs by use of a cardiomyogenic differentiation medium, which is a combination of several agents including hormones (e.g., insulin); growth factors (e.g., bFGF and IGF); serum and other agents such as immuno-suppressing agent (e.g., dexamethasone and LIF) and vitamin (e.g., ascorbic acid). There is also a publication that teaches the use of a more simplified cardiomyogenic differentiation medium, however, this differentiation medium was used to differentiate embryonic stem cells (ESCs), instead of bone marrow mesenchymal stem cells. For example, Takahashi et al (Takahashi et al., (2003) Circulation, 107:1912-1916) disclosed the induction of differentiation of ESCs into cardiomyocytes by 0.01 mM ascorbic acid (see FIG. 2 of Takahashi et al), and this effect of ascorbic acid is not mimicked by the other antioxidants such as N-acetylcysteine, Tiron or vitamin E (see FIG. 5 of Takahashi et al). However, the role of ascorbic acid in induction of cardiac differentiation of ESCs is still controversial. Another publication, WO 2005/065354, disclosed the use of a defined medium in maintaining the undifferentiated growth of human ESCs, said medium comprises sufficient amounts of bFGF, insulin, and ascorbic acid (see claim 1 of '354 patent). Example 8 of '354 patent, which is directed to a method of inducing cardia differentiation, was accomplished by culturing the undifferentiated human ESCs in a standard differentiation media consisting of KO-DMEM (80%), defined FBS (20%), L-glutamine (2 mM), MEM nonessential amino acids (1×) and β-mercaptoethanol (100 μM); and beating cardiomyocytes were observed in about 1 week, increased in numbers with time, and retained contractility for over two months. In other words, Example 8 of '354 patent implicates that the induction of human ESCs into cardiomyocytes may be achieved in the absence of ascorbic acid.
In view of the above, to date, there isn't any publication discloses the use of ascorbic acid in induction of differentiation of bone marrow mesenchymal stem cells into cardiomyocytes, and there exists in this art a need of an improved method of producing cardiomyocytes from bone marrow mesenchymal stem cells, such method is simple to use and with high producing rate that large amount of differentiated cardiomyocytes may be obtained in a relatively short period of time to ensure a successful cell-based therapy.